Towards Practical Application of Methanotrophic Metabolism in Chlorinated Hydrocarbon Degradation, Greenhouse Gas Removal, and Immobilization of Heavy Metals.

Methanotrophs are a group of microorganisms found in diverse environments that utilize methane for energy and biomass. These microorganisms have long been studied for their practical applications as well as for their significant contributions to the global carbon and nitrogen cycles. The scope of this study was to closely examine the physiological and genetic properties of different groups of methanotrophs to the utility of these cells for: (1) pollutant degradation by these cells and (2) removal of methane from the atmosphere. Finally, a new assay was created to screen methanotrophs for production of methanobactin, a unique copper chelating biomolecule that has significant potential industrial applications engineering practices or development of biological engineering devices and processes de novo utilizing these bacteria. Biodegradation of TCE, trans-DCE, and VC by methanotrophs was closely examined in this study. Significance of selective expression of sMMO and pMMO in biodegradation activity of Methylosinus trichosporium OB3b was examined at a realistic in situ groundwater temperature condition to support the argument that pMMO is responsible for methane-augmented bioremediation. Also Methylocystis daltona SB2 was examined for the expression of pMMO and degradation of chlorinated ethene compounds under growth on acetate in absence of methane using quantitative RT–PCR technique. Considering the significant contribution of methane emission to the greenhouse effect, the ability of methanotrophs to use methane as the electron donor has great significance in environmental perspective. In this study, a methanotrophic biotrickling filtration system was designed to actively utilize methanotrophic activity to remove atmospheric methane from local methane “hotspots”. Mathematical modeling was performed to assess the technological and economical feasibility of this biofiltration system. Methanobactin, a copper chelating biomolecule, has been hypothesized to have significant roles in methanotrophy, as well as interesting potential applications. To enable the genetic studies on methanobactin, we have adopted CAS assay for screening methanotrophs for production of methanobactin. The assay was successful in screening the methanotrophic strains for differential capability to abstract copper. Altogether, we expect the findings in this study to serve as basis for further research that will allow better understanding and more effective applications of methanotrophs.Ph.D.Environmental EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77901/1/yunsh_1.pd

Measurement and modeling of multiple substrate oxidation by methanotrophs at 20 °C

Earlier experiments have shown that when Methylosinus trichosporium OB3b was grown at 30 °C, greater growth and degradation of chlorinated ethenes was observed under particulate methane monooxygenase (pMMO)-expressing conditions than sMMO-expressing conditions. The effect of temperature on the growth and ability of methanotrophs to degrade chlorinated ethenes, however, has not been examined, particularly temperatures more representative of groundwater systems. Thus, experiments were performed at 20 °C to examine the effect of mixtures of trichloroethylene, trans -dichloroethylene and vinyl chloride in the presence of methane on the growth and ability of Methylosinus trichosporium OB3b cells to degrade these pollutants. Although the maximal rates of chlorinated ethane degradation were greater by M. trichosporium OB3b expressing sMMO as compared with the same cell expressing pMMO, the growth and ability of sMMO-expressing cells to degrade these cosubstrates was substantially inhibited in their presence as compared with the same cell expressing pMMO. The Δ model developed earlier was found to be useful for predicting the effect of chlorinated ethenes on the growth and ability of M. trichosporium OB3b to degrade these compounds at a growth temperature of 20 °C. Finally, it was also discovered that at 20 °C, cells expressing pMMO exhibited faster turnover of methane than sMMO-expressing cells, unlike that found earlier at 30 °C, suggesting that temperature may exert selective pressure on methanotrophic communities to express sMMO or pMMO.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75464/1/j.1574-6968.2008.01314.x.pd

Methanotrophs and copper

Methanotrophs, cells that consume methane (CH 4 ) as their sole source of carbon and energy, play key roles in the global carbon cycle, including controlling anthropogenic and natural emissions of CH 4 , the second-most important greenhouse gas after carbon dioxide. These cells have also been widely used for bioremediation of chlorinated solvents, and help sustain diverse microbial communities as well as higher organisms through the conversion of CH 4 to complex organic compounds (e.g. in deep ocean and subterranean environments with substantial CH 4 fluxes). It has been well-known for over 30 years that copper (Cu) plays a key role in the physiology and activity of methanotrophs, but it is only recently that we have begun to understand how these cells collect Cu, the role Cu plays in CH 4 oxidation by the particulate CH 4 monooxygenase, the effect of Cu on the proteome, and how Cu affects the ability of methanotrophs to oxidize different substrates. Here we summarize the current state of knowledge of the phylogeny, environmental distribution, and potential applications of methanotrophs for regional and global issues, as well as the role of Cu in regulating gene expression and proteome in these cells, its effects on enzymatic and whole-cell activity, and the novel Cu uptake system used by methanotrophs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79061/1/j.1574-6976.2010.00212.x.pd

Strategies to Optimize Microbially-Mediated Mitigation of Greenhouse Gas Emissions from Landfill Cover Soils

The overall objective of this project, 'Strategies to Optimize Microbially-Mediated Mitigation of Greenhouse Gas Emissions from Landfill Cover Soils' was to develop effective, efficient, and economic methodologies by which microbial production of nitrous oxide can be minimized while also maximizing microbial consumption of methane in landfill cover soils. A combination of laboratory and field site experiments found that the addition of nitrogen and phenylacetylene stimulated in situ methane oxidation while minimizing nitrous oxide production. Molecular analyses also indicated that methane-oxidizing bacteria may play a significant role in not only removing methane, but in nitrous oxide production as well, although the contribution of ammonia-oxidizing archaea to nitrous oxide production can not be excluded at this time. Future efforts to control both methane and nitrous oxide emissions from landfills as well as from other environments (e.g., agricultural soils) should consider these issues. Finally, a methanotrophic biofiltration system was designed and modeled for the promotion of methanotrophic activity in local methane 'hotspots' such as landfills. Model results as well as economic analyses of these biofilters indicate that the use of methanotrophic biofilters for controlling methane emissions is technically feasible, and provided either the costs of biofilter construction and operation are reduced or the value of CO{sub 2} credits is increased, can also be economically attractive

Novel copper-containing membrane monooxygenases (CuMMOs) encoded by alkane-utilizing Betaproteobacteria.

Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short-chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived protein sequence identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell amplified genomes (SAGs) from propane-enriched samples were screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium encoded enzymes with the potential to support catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies these enzymes in non-nitrifying Betaproteobacteria

pH Control Enables Simultaneous Enhancement of Nitrogen Retention and N2O Reduction in Shewanella loihica Strain PV-4

pH has been recognized as one of the key environmental parameters with significant impacts on the nitrogen cycle in the environment. In this study, the effects of pH on NO3–/NO2– fate and N2O emission were examined with Shewanella loihica strain PV-4, an organism with complete denitrification and respiratory ammonification pathways. Strain PV-4 was incubated at varying pH with lactate as the electron donor and NO3–/NO2– and N2O as the electron acceptors. When incubated with NO3– and N2O at pH 6.0, transient accumulation of N2O was observed and no significant NH4+ production was observed. At pH 7.0 and 8.0, strain PV-4 served as a N2O sink, as N2O concentration decreased consistently without accumulation. Respiratory ammonification was upregulated in the experiments performed at these higher pH values. When NO2– was used in place of NO3–, neither growth nor NO2– reduction was observed at pH 6.0. NH4+ was the exclusive product from NO2– reduction at both pH 7.0 and 8.0 and neither production nor consumption of N2O was observed, suggesting that NO2– regulation superseded pH effects on the nitrogen-oxide dissimilation reactions. When NO3– was the electron acceptor, nirK transcription was significantly upregulated upon cultivation at pH 6.0, while nrfA transcription was significantly upregulated at pH 8.0. The highest level of nosZ transcription was observed at pH 6.0 and the lowest at pH 8.0. With NO2– as the electron acceptor, transcription profiles of nirK, nrfA, and nosZ were statistically indistinguishable between pH 7.0 and 8.0. The transcriptions of nirK and nosZ were severely downregulated regardless of pH. These observations suggested that the kinetic imbalance between N2O production and consumption, but neither decrease in expression nor activity of NosZ, was the major cause of N2O accumulation at pH 6.0. The findings also suggest that simultaneous enhancement of nitrogen retention and N2O emission reduction may be feasible through pH modulation, but only in environments where C:N or NO2–:NO3– ratio does not exhibit overarching control over the NO3–/NO2– reduction pathways

Design and Feasibility Analysis of a Self-Sustaining Biofiltration System for Removal of Low Concentration N₂O Emitted from Wastewater Treatment Plants

N₂O is a potent greenhouse gas and ozone-depletion agent. In this study, a biofiltration system was designed for removal of N₂O emitted at low concentrations (<200 ppmv) from wastewater treatment plants. The proposed biofiltration system utilizes untreated wastewater from the primary sedimentation basin as the source of electron donor and nutrients and energy requirement is minimized by utilizing gravitational force and pressure differential to direct liquid medium and gas through the biofilter. The experiments performed with laboratory-scale biofilter in two different configurations confirmed the feasibility of the biofiltration system. The biofilter operated with cycling of raw wastewater exhibited up to 94% and 53% removal efficiency with 100 ppmv N₂O in N₂ and air, respectively, as the feed gas, corroborating that untreated wastewater can serve as a robust source of electron donor and nutrients. The laboratory-scale biofilter operated with a continuous flow-through of synthetic wastewater attained >99.9% removal of N₂O from N₂ background at the gas flow rate up to 2,000 mL·min^–1 and >50% N₂O removal from air background at the gas flow rate of 200 mL·min^–1. <i>nosZ</i>-containing bacterial genera including <i>Flavobacterium</i> (5.92%), <i>Pseudomonas</i> (4.26%) and <i>Bosea</i> (2.39%) were identified in the biofilm samples collected from the oxic biofilter, indicating these organisms were responsible for N₂O removal

An assay for screening microbial cultures for chalkophore production

Methanotrophs, bacteria that utilize methane as their sole carbon and energy source, are known to have high requirements for copper. These bacteria have recently been found to synthesize a copper-chelating agent, or chalkophore, termed methanobactin. To aid in screening methanobactin production by methanotrophs, a plate assay developed from the chrome azurol S (CAS) assay for siderophore production, was modified. In the typical CAS assay, a colour change from blue to orange in iron–CAS plates is observed as iron (III) ion weakly bound to CAS is sequestered by siderophores with higher affinities. In our modified assay, iron (III) chloride of the original CAS solution was substituted with copper (II) chloride, and removal of copper from CAS caused a colour change from blue to yellow. Assay results indicated that of the four tested methanotrophs ( Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomicrobium album BG8 and Methylocystis parvus OBBP), only M. trichosporium OB3b, M. capsulatus Bath and M. album BG8 produced chalkophores capable of competing with CAS for copper, while M. parvus OBBP did not or did not export sufficient concentrations of methanobactin for detection by this assay. It was also found using Fe–CAS plates that at least M. trichosporium OB3b and M. album BG8 produce siderophores. These results may be expanded for the detection of chalkophores in other microorganisms as well as for screening of putative mutants of chalkophore synthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78618/1/EMI4_125_sm_Fig_S2.pd

Strategies to Optimize Microbially-Mediated Mitigation of Greenhouse Gas Emissions from Landfill Cover Soils

The overall objective of this project, 'Strategies to Optimize Microbially-Mediated Mitigation of Greenhouse Gas Emissions from Landfill Cover Soils' was to develop effective, efficient, and economic methodologies by which microbial production of nitrous oxide can be minimized while also maximizing microbial consumption of methane in landfill cover soils. A combination of laboratory and field site experiments found that the addition of nitrogen and phenylacetylene stimulated in situ methane oxidation while minimizing nitrous oxide production. Molecular analyses also indicated that methane-oxidizing bacteria may play a significant role in not only removing methane, but in nitrous oxide production as well, although the contribution of ammonia-oxidizing archaea to nitrous oxide production can not be excluded at this time. Future efforts to control both methane and nitrous oxide emissions from landfills as well as from other environments (e.g., agricultural soils) should consider these issues. Finally, a methanotrophic biofiltration system was designed and modeled for the promotion of methanotrophic activity in local methane 'hotspots' such as landfills. Model results as well as economic analyses of these biofilters indicate that the use of methanotrophic biofilters for controlling methane emissions is technically feasible, and provided either the costs of biofilter construction and operation are reduced or the value of CO{sub 2} credits is increased, can also be economically attractive